Journal of Steroid Biochemistry & Molecular Biology 75 (2000) 291 – 298 Expression of cytochrome P450 11 (11-hydroxylase) gene during gonadal sex differentiation and spermatogenesis in rainbow trout, Oncorhynchus mykiss Shaojun Liu a,1 , Marina Govoroun a , Helena D’Cotta b , Marie-Jose ´ Ricordel a , Jean-Jacques Lareyre a , Oonagh Marie McMeel c , Terry Smith c , Yoshitaka Nagahama d , Yann Guiguen a, * a Station INRA SCRIBE, Equipe Sexualite et Reproduction, Campus de Beaulieu, 35042 Rennes Cedex, France b CIRAD-EMVT Campus de Beaulieu, 35042 Rennes Cedex, France c National Diagnostic Centre (NDC), National Uniersity of Ireland, Uniersity Road, Galway, Ireland d Laboratory of Reproductie Biology, National Institute for Basic Biology, Okazaki 444, Japan Accepted 20 October 2000 Abstract Androgens and especially 11-oxygenated androgens are known to be potent masculinizing steroids in fish. As a first step to study their physiological implication in gonadal sex differentiation in fish, we cloned a testicular cytochrome P450 11 (11-hydrox- ylase) cDNA in the rainbow trout, Oncorhynchus mykiss. We isolated a 1882 bp P450 11 cDNA (rt11H2, AF217273) which contains an open reading frame encoding a 552 putative amino acids protein. This sequence was highly homologous (98% in nucleotides and 96.5% in amino acids) to another rainbow trout P450 11 sequence (AF179894) and also to a Japanese eel P450 11 (68% in amino acids). Northern blot analysis detected a single transcript of 2 kb which was highly expressed in the testis (stage II) and to a lesser degree in the anterior kidney (containing the interrenal tissue). No signal was detected in the posterior kidney, brain, liver, skin, intestine and heart. In the testis this transcript was highly expressed at the beginning of spermatogenesis (stages I and II), followed by a decrease during late spermatogenesis (stages III to V). By semi-quantitative reverse transcription polymerase chain reaction, P450 11 expression during gonadal differentiation was estimated to be at least 100 times higher in male than in female gonads. This difference was first detected at 55 days post-fertilization (dpf), i.e. 3 weeks before the first sign of histological sex differentiation, and was sustained long after differentiation (127 dpf). Specific P450 11 gene expression was also demonstrated before testis differentiation (around 50 dpf) using virtual Northern blot, with no expression detected in female differentiating gonads. From these results, and also based on the already known actions of 11-oxygenated androgens in testicular differentiation in fish, it is now suggested that P450 11 gene expression is a key factor for the testicular differentiation in rainbow trout. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Fish; Steroids; 11-Oxygenated androgens; Testis; Interrenal www.elsevier.com/locate/jsbmb 1. Introduction Steroids have been shown to play a crucial role in non-mammalian vertebrate gonadal sex differentiation in fish [1], amphibian [2], reptiles [3,4] and birds [5,6]. According to Yamamoto [7], androgens in fish would act as andro-inducers driving testis differentiation and estrogens as gyno-inducers driving ovarian differentia- tion. This model was primarily based on the masculiniz- ing and feminizing effects of androgens and estrogens, Abbreiations: bp, base pair; 11H, 11-hydroxylase; dpf, days post fertilization; kb, kilobase; RT-PCR, reverse transcription polymerase chain reaction. * Corresponding author. Tel.: +33-2-23485009; fax: +33-2- 23485020. E-mail address: guiguen@beaulieu.rennes.inra.fr (Y. Guiguen). 1 Present address: Department of Biology, Hunan Normal Univer- sity, Changsha, Hunan, People’s Republic of China. 0960-0760/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII:S0960-0760(00)00186-2